Dysregulation of the phosphoinositide 3-kinase (PI3K) pathway is implicated in many human diseases. Hyperactivation of this pathway contributes to human cancers, and defects in the pathway play a role in the development of type II diabetes. Therefore, key steps in this pathway represent promising targets for the development of drugs to combat these and other diseases associated with dysregulation of the PI3K pathway.
Class I PI3Ks (α, β and γ) are recruited to the plasma membrane in response to growth factor and hormone stimulation, where catalytic p110 subunits phosphorylate lipid phosphatidylinositol-4,5-bisphosphate (PIP2) at the D3 position to generate a second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3). Cantley L C (2002) Science 296:1655-7. Conversely, the phosphatase PTEN specifically dephosphorylates the D3 position of PIP3 to produce PIP2. Loss of PTEN protein expression or function has been found in a large fraction of the advanced human cancers, indicating that uncontrolled signaling through PI3K contributes to tumorigenesis and metastasis. Maehama T et al. (1999) Trends Cell Biol 9:125-8. Furthermore, transgenic studies directly established that loss of PTEN leads to tumorigenesis. Li J et al. (1997) Science 275:1943-7; Vivanco I et al. (2002) Nat Rev Cancer 2:489-501.
PIP3 controls a complex cellular signaling network regulating cell growth, proliferation and cell survival, cytoskeletal rearrangements, and cell migration. PIP3 target proteins are located in the cytosol of unstimulated cells, but in response to lipid phosphorylation translocate to the plasma membrane because of their ability to associate with the newly formed PIP3 through its direct binding to pleckstrin-homology (PH) domains. Membrane translocation and, in some cases, activation of the PIP3 target factors initiate a variety of local responses, including polymerization of actin, assembly of signaling complexes, and priming of protein kinase cascades. Cantley L C (2002) Science 296:1655-7; Fruman D A et al. (1998) Annu Rev Biochem 67:481-507; Vivanco I et al. (2002) Nat Rev Cancer 2:489-501.
Among the PIP3-controlled signaling proteins, the serine-threonine Akt/PKB (Akt) protein kinase family is of particular interest, because it has been found to play a central role in wide range of fundamental cellular functions including cell survival, growth, and energy metabolism. Datta S R et al. (1999) Genes Dev 13:2905-27; Scheid M P et al. (2001) Nat Rev Mol Cell Biol 2:760-8. The mechanism by which Akt protects cells from death is likely to be multifactorial, involving direct phosphorylation of multiple components of the cell-death machinery such as FOXO transcription factors, BAD, glycogen synthase kinase-3 (GSK-3), and caspase-9. Akt also enhances protein synthesis and cell growth by activating mammalian target of rapamycin (mTOR), which, in concert with another PIP3-binding kinase, PDK1, stimulates p70 ribosomal protein S6 kinase (p70S6K) and inhibits translational repressor eukaryotic initiation factor 4E-binding protein 1 (4EBP1). Cardone M H et al. (1998) Science 282:1318-21; Cross D A et al. (1995) Nature 378:785-9; Datta S R et al. (1997) Cell 91:231-241; McManus E J et al. (2002) Nat Cell Biol 4:E214-216; Tee A R et al. (2002) Proc Natl Acad Sci USA 99:13571-6.
Other PH domain-containing proteins that are activated by PIP3 include general receptor for phosphoinositides-1 (GRP1, also known as cytohesin-3), GDP/GTP exchange factors (GEFs) for Rac, Rho and Cdc42 GTPases and ADP-ribosylating factor 6 (ARF6) and protein tyrosine kinases (PTKs) of the Bruton's tyrosine kinase (Btk) and Tec families. Venkateswarlu K et al. (1998) Biochem J 335:3139-46; Han J et al. (1998) Science 279:558-60; Salim K et al. (1996) EMBO J. 15:6241-50; Venkateswarlu K et al. (2000) Biochem J 345 Pt 3:719-24. Activation of the Rac family or ARF6 by local gradients of PIP3 plays a major role in remodeling the actin cytoskeleton for directional motility in response to chemotactic agents and growth stimulation. These mechanisms play an important role in enhanced motility of cancer cells and cancer metastasis. Etienne-Manneville S et al. (2002) Nature 420:629-35; Hornstein I et al. (2004) Cell Signal 16:1-11; Venkateswarlu K et al. (2000) Biochem J 345 Pt 3:719-24.
Lipid-protein interactions play a key role in the downstream signaling in the PI3K pathway. Furthermore, this step is most commonly deregulated in cancer cells due to the loss of PTEN. However, until now, most of the therapeutic strategies targeting the PI3K pathway have focused on the development of inhibitors of PI3K, PDK1, or Akt kinase. Peifer C et al. (2008) ChemMedChem 3:1810-38; Powis G et al. (1994) Cancer Res 54:2419-23; Vlahos C J et al. (1994) J Biol Chem 269:5241-8; Yaguchi S et al. (2006) J Natl Cancer Inst 98:545-56; Yang L et al. (2004) Cancer Res 64:4394-9. Inhibitors of protein-protein interactions as cell biology tools and leads for drug development have attracted interest in recent years due to the recognition of the key role of such interactions in cellular signaling. Phospholipid-protein interactions, however, have not been as actively targeted, even though lipid molecules represent one of the most important classes of second messengers. This is surprising considering that they represent “prototypic” small molecule-protein interactions usually involving well defined binding sites. Lemmon M A (2008) Nat Rev Mol Cell Biol 9:99-111. Therefore, protein-lipid interactions may be more readily targetable by chemical inhibitors compared to protein-protein interactions, the latter frequently involving binding of the extended flat protein surfaces, thus presenting significant challenge for disruption by small molecules.
Degterev et al. recently identified a new class of non-lipid small molecule inhibitors targeting a broad range of PIP3-dependent signaling events in vitro and possessing significant anti-tumor activity in vivo. WO 2011/022028, filed May 17, 2010. This class of molecules includes compounds of Formula I
wherein X is selected from the group consisting of H and Cl, and each of R1 and R2 is independently selected from the group consisting of H and CH3. These molecules were found to reduce binding of PIP3 to pleckstrin homology domain, thereby inhibiting PIP3-dependent cellular signaling pathways in a cell.